The aim of this study was to investigate the relationship between opium and amphetamine dependency with the serum melatonin levels in the presence of circadian rhythm sleep disorders (CRSD). Forty four male amphetamine-dependent and opium-dependent patients with CRSD and with more than one year substance dependency were enrolled in this study. Control group consisted of twelve healthy male subjects. The diagnoses of sleep disorders were established by a psychiatrist and were made on the basis of the criteria of ICSD-II using the patients' sleep logs. Blood samples were drawn every 4 h through an intravenous catheter. Serum melatonin levels were assayed using an enzyme-linked immunosorbent assay (ELISA) kit. Repeated Measures Analysis of variance (ANOVA) was used to assess differences between the melatonin levels at six separate times. The serum melatonin levels of the control subjects were significantly higher than both opium-dependent and amphetamine-dependent patients at 24:00, 4:00 and 8:00. The serum melatonin level of the opium-dependent patients were significantly lower than the amphetamine-dependent patients at 24:00 (26.9 ± 11.4 vs. 41 ± 19.4, respectively; p = 0.006) and were significantly higher than the amphetamine-dependent patients at 16:00 (12.7 ± 5.1 vs. 8.9 ± 4.1, respectively; p = 0.011). This is an evidence of negative effects of substance dependence on circadian cycle of melatonin secretion among opium and amphetamine dependent patients.